Engineering Allosteric Ribozymes to Detect Thiamine Pyrophosphate in Whole Blood

Thiamine deficiency contributes to several human diseases including Alzheimer's. As its biologically active form, thiamine pyrophosphate (TPP) has been considered as a potential biomarker for Alzheimer's disease (AD) based on several clinical reports that apparently lower blood TPP levels were found in patients with mild cognitive impairment to AD. However, highly sensitive and high-throughput detection of TPP in biological fluids remains an analytical challenge. Here, we report engineering RNA-based sensors to quantitatively measure TPP concentrations in whole blood samples with a detection limit down to a few nM. By fusing a TPP-specific aptamer with the hammerhead ribozyme for in vitro selection, we isolated an allosteric ribozyme with an EC₅₀ value (68 nM) similar to the aptamer's K_D value (50 nM) for TPP, which for the first time demonstrates the possibility to maintain the effector binding affinity of the aptamer in such engineered allosteric RNA constructs. Meanwhile, we developed a new blood sample preparation protocol to be compatible with RNA. By coupling the TPP-induced ribozyme cleavage event with isothermal amplification, we achieved fluorescence monitoring of whole blood TPP levels through the "mix-and-read" operation with high-throughput potential. We expect that the engineered TPP-sensing RNAs will facilitate clinical research on AD as well as other thiamine-related diseases.